NDLI: Mutational profiling of micro-dissected pre-malignant lesions from archived specimens

Content Provider	Springer Nature : BioMed Central
Author	Nachmanson, Daniela Steward, Joseph Yao, Huazhen Officer, Adam Jeong, Eliza O’Keefe, Thomas J. Hasteh, Farnaz Jepsen, Kristen Hirst, Gillian L. Esserman, Laura J. Borowsky, Alexander D. Harismendy, Olivier
Abstract	Background Systematic cancer screening has led to the increased detection of pre-malignant lesions (PMLs). The absence of reliable prognostic markers has led mostly to over treatment resulting in potentially unnecessary stress, or insufficient treatment and avoidable progression. Importantly, most mutational profiling studies have relied on PML synchronous to invasive cancer, or performed in patients without outcome information, hence limiting their utility for biomarker discovery. The limitations in comprehensive mutational profiling of PMLs are in large part due to the significant technical and methodological challenges: most PML specimens are small, fixed in formalin and paraffin embedded (FFPE) and lack matching normal DNA. Methods Using test DNA from a highly degraded FFPE specimen, multiple targeted sequencing approaches were evaluated, varying DNA input amount (3–200 ng), library preparation strategy (BE: Blunt-End, SS: Single-Strand, AT: A-Tailing) and target size (whole exome vs. cancer gene panel). Variants in high-input DNA from FFPE and mirrored frozen specimens were used for PML-specific variant calling training and testing, respectively. The resulting approach was applied to profile and compare multiple regions micro-dissected (mean area 5 mm2) from 3 breast ductal carcinoma in situ (DCIS). Results Using low-input FFPE DNA, BE and SS libraries resulted in 4.9 and 3.7 increase over AT libraries in the fraction of whole exome covered at 20x (BE:87%, SS:63%, AT:17%). Compared to high-confidence somatic mutations from frozen specimens, PML-specific variant filtering increased recall (BE:85%, SS:80%, AT:75%) and precision (BE:93%, SS:91%, AT:84%) to levels expected from sampling variation. Copy number alterations were consistent across all tested approaches and only impacted by the design of the capture probe-set. Applied to DNA extracted from 9 micro-dissected regions (8 PML, 1 normal epithelium), the approach achieved comparable performance, illustrated the data adequacy to identify candidate driver events (GATA3 mutations, ERBB2 or FGFR1 gains, TP53 loss) and measure intra-lesion genetic heterogeneity. Conclusion Alternate experimental and analytical strategies increased the accuracy of DNA sequencing from archived micro-dissected PML regions, supporting the deeper molecular characterization of early cancer lesions and achieving a critical milestone in the development of biology-informed prognostic markers and precision chemo-prevention strategies.
Related Links	https://bmcmedgenomics.biomedcentral.com/counter/pdf/10.1186/s12920-020-00820-y.pdf
Ending Page	15
Page Count	15
Starting Page	1
File Format	HTM / HTML
ISSN	17558794
DOI	10.1186/s12920-020-00820-y
Journal	BMC Medical Genomics
Issue Number	1
Volume Number	13
Language	English
Publisher	BioMed Central
Publisher Date	2020-11-18
Access Restriction	Open
Subject Keyword	Human Genetics Microarrays Gene Expression Targeted sequencing Cancer progression Pre-malignant lesion Variant calling FFPE Micro-dissection Breast DCIS
Content Type	Text
Resource Type	Article
Subject	Genetics (clinical) Genetics
Journal Impact Factor	2.1/2023
5-Year Journal Impact Factor	2.5/2023

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